This invention relates generally to the handling of bead-based combinatorial libraries, and more particularly to an improved process and apparatus for depositing beads in two-dimensional arrays. This invention also relates to an improved means of synthesizing combinatorial libraries in an array format.
Synthesis of combinatorial compound libraries on bead supports is a well-established method for generating chemical diversity for screening against targets of pharmacological relevance. Such libraries may be synthesized as bulk populations or discrete sublibraries with or without identifier tags for deconvolution. In most cases each bead carries a single unique compound and is present in a mixture of beads containing other compounds. To test the beads for activity against a pharmacological target, it is desirable to separate the beads so that each bead may be individually assayed against one or more targets. The beads carrying active compounds can be identified and the structure of the compound elucidated. It is also desirable to separate the beads into arrays that are geometrically compatible with robotic screening systems, for example 12xc3x978 arrays or other formats such as 384 or 1,536-well configurations. An alternative approach to the synthesis and evaluation of bead-based libraries is to array the beads prior to synthesis of the library components. This approach has the advantage that the synthetic history of each component is known from its position in the array, but has the disadvantage that the synthesis process itself is less facile, particularly for large libraries. A process and apparatus that can be used for either approach would represent a substantial improvement over current practice.
Current methods of bead arraying including manual picking and hydrodynamic sorting, in which beads are allowed to flow though an aperture and once detected are deposited into assay wells. Manual picking is extremely slow and tedious. Hydrodynamic methods sort in serial mode and are therefore slow and prone to clogging. Further, buoyancy variations within the bead library are known to cause problems. Therefore there is a need for a simple, rapid method for bead arraying which reliably delivers single beads, or a predetermined desired number of beads to each point in the array, which is not affected by the presence of compound on the beads, and which can be configured to suit any desirable format.
Based on the foregoing, it can be seen that there is a pressing need in the art for improvements in bead arraying equipment and methods. The principal object of this invention, therefore, is to provide an improved method for arraying beads, whether or not they have compounds attached to them. Another object of the invention is to provide an improved method for delivery of beads to a desired geometric array. It is also an object of the invention to provide an improved apparatus for bead arraying which is inexpensive, easy to use and independent of the presence and identity of compounds on the beads. It is a further object of this invention to provide an improved apparatus for synthesis of bead-based combinatorial libraries in array format, such apparatus being compatible with the manipulation of beads that already have compounds attached.
In accordance with the invention, the arraying of beads for bead-based combinatorial libraries is carried out by establishing a bead-receiving surface comprising two components: a pattern of discrete bead attachment areas, capable of holding a limited number of beads of uniform size; and an area, from which beads are free to separate, spacing the discrete bead attachment areas from one another. The limited number of beads which each bead attachment area is capable of holding can be one or more than one, and is preferably the same as that for every other bead attachment area in the pattern. A mixture of beads of uniform size is placed onto the bead-receiving surface, and, preferably by agitating the bead mixture, a predetermined number of beads from the mixture is caused to attach to each of the discrete bead attachment areas. Beads not attached to the discrete bead attachment areas are removed. Then the attached beads, which are in a pattern corresponding to the pattern of the bead attachment areas are released and deposited into any array.
In one embodiment of the invention, each discrete bead attachment area is a portion of a continuous adhesive layer exposed through an aperture in a mask. The area from which the beads are free to separate is a surface of the mask. In another embodiment, the step of establishing a bead-receiving surface is carried out by applying minute adhesive droplets to a substrate in a pattern.
The step of releasing the attached beads is preferably preceded by the step of aligning the attached beads with receptacles in an array corresponding to the pattern of bead attachment areas, and the depositing step is carried out by depositing the beads into the receptacles with which they are aligned.
Where a porous substrate is used, and the bead attachment areas are areas of adhesive on the substrate, the attached beads can be released by washing the substrate with a solvent and thereby dissolving the bead attachment areas.
In the case of beads having attached compounds representing a combinatorial library, the depositing step is preferably followed by the step of cleaving a compound from each bead by means of a reagent, while the beads remain in the receptacle. The receptacles are preferably in the form of glass capillaries arranged so that they can be simultaneously immersed into reagents in the wells of a well plate after the depositing step is carried out. Compounds can be cleaved from the beads by the reagents in the wells while the beads remain in the capillaries, and the cleaved compounds can then be tested for pharmaceutical activity.
Synthesis of chemical compounds on bead arrays can be carried out using a similar procedure. The process for synthesis of pre-arrayed libraries according to the invention is carried out by arraying blank beads into capillaries. The capillaries containing the beads are dipped into multiwell containers, each well containing a different chemical reagent. Compounds are assembled on the beads by successive transfer of the capillary into appropriate reagent plates. Once complete, the compounds are released from the beads by transfer of the capillary array into a receptacle containing cleavage reagents.
A synthesizing process in accordance with the invention comprises the steps of: establishing a bead-receiving surface comprising a pattern of discrete bead attachment areas, each capable of holding a limited number of beads of uniform size and an area from which beads are free to separate, spacing the discrete bead attachment areas from one another. Here also, the limited number of beads for each bead attachment area is preferably the same as that for every other bead attachment area in the pattern. A mixture of beads of uniform size is placed onto the bead-receiving surface, and a predetermined number of beads from the mixture is caused to attach to each of the discrete bead attachment areas. Unattached beads are removed, and the attached beads are aligned with capillaries in an array of capillaries corresponding to the pattern of bead attachment areas. The attached beads are then released and deposited into the array of capillaries. The capillaries of the array are introduced simultaneously into wells containing reagents for desired synthetic steps, and syntheses are carried out on the beads while the beads remain in the capillaries. The synthesis may be followed by washing the beads in the capillaries to remove unbound reagents, and cleaving synthesized compounds.
Another feature of the invention resides in the structure of the apparatus used in the case in which the bead attachment areas are portions of a continuous adhesive layer. The apparatus comprises a mask and substrate. The mask is preferably the floor of a tray and has parallel first and second faces and an array of apertures, each aperture extending from the first face to the second face. The apertures are of uniform size such that he number of beads that can be located abreast in each aperture is limited to a predetermined number. At least in the case in which each aperture is intended to receive only one bead, the apertures are preferably circular. The substrate is preferably a porous sheet of nitrocellulose removable from the mask, and has a face with a layer of adhesive, preferably ethylene glycol, situated against the second face of the mask and covering openings of the apertures in the second face. Thus, a predetermined number of beads, at each aperture, can be deposited in the second face. Thus, a predetermined number of beads, at each aperture, can be deposited onto, and adhere to, the adhesive layer. Means are preferably provided for holding the mask in a fixed relationship with an array of receptacles, so that beads deposited through the apertures of the mask onto the adhesive layer of the substrate can be readily aligned with the receptacles.
Various other objects, details and advantages of the invention will be apparent from the following detailed description, when read in conjunction with the drawings: